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  • 1.
    Abelein, Axel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Abrahams, Jan Pieter
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jarvet, Juri
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. National Institute of Chemical Physics and Biophysics, Estonia.
    Luo, Jinghui
    Tiiman, Ann
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wärmländer, Sebastian K. T. S.
    The hairpin conformation of the amyloid beta peptide is an important structural motif along the aggregation pathway2014In: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 19, no 4-5, p. 623-634Article, review/survey (Refereed)
    Abstract [en]

    The amyloid beta (A beta) peptides are 39-42 residue-long peptides found in the senile plaques in the brains of Alzheimer's disease (AD) patients. These peptides self-aggregate in aqueous solution, going from soluble and mainly unstructured monomers to insoluble ordered fibrils. The aggregation process(es) are strongly influenced by environmental conditions. Several lines of evidence indicate that the neurotoxic species are the intermediate oligomeric states appearing along the aggregation pathways. This minireview summarizes recent findings, mainly based on solution and solid-state NMR experiments and electron microscopy, which investigate the molecular structures and characteristics of the A beta peptides at different stages along the aggregation pathways. We conclude that a hairpin-like conformation constitutes a common motif for the A beta peptides in most of the described structures. There are certain variations in different hairpin conformations, for example regarding H-bonding partners, which could be one reason for the molecular heterogeneity observed in the aggregated systems. Interacting hairpins are the building blocks of the insoluble fibrils, again with variations in how hairpins are organized in the cross-section of the fibril, perpendicular to the fibril axis. The secondary structure propensities can be seen already in peptide monomers in solution. Unfortunately, detailed structural information about the intermediate oligomeric states is presently not available. In the review, special attention is given to metal ion interactions, particularly the binding constants and ligand structures of A beta complexes with Cu(II) and Zn(II), since these ions affect the aggregation process(es) and are considered to be involved in the molecular mechanisms underlying AD pathology.

  • 2.
    Abelein, Axel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bolognesi, Benedetta
    Dobson, Christopher M.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lendel, Christofer
    Hydrophobicity and conformational change as mechanistic determinants for nonspecific modulators of amyloid β self-assembly2012In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, no 1, p. 126-137Article in journal (Refereed)
    Abstract [en]

    The link between many neurodegenerative disorders, including Alzheimer's and Parkinson's diseases, and the aberrant folding and aggregation of proteins has prompted a comprehensive search for small organic molecules that have the potential to inhibit such processes. Although many compounds have been reported to affect the formation of amyloid fibrils and/or other types of protein aggregates, the mechanisms by which they act are not well understood. A large number of compounds appear to act in a nonspecific way affecting several different amyloidogenic proteins. We describe here a detailed study of the mechanism of action of one representative compound, lacmoid, in the context of the inhibition of the aggregation of the amyloid β-peptide (Aβ) associated with Alzheimer's disease. We show that lacmoid binds Aβ(1-40) in a surfactant-like manner and counteracts the formation of all types of Aβ(1-40) and Aβ(1-42) aggregates. On the basis of these and previous findings, we are able to rationalize the molecular mechanisms of action of nonspecific modulators of protein self-assembly in terms of hydrophobic attraction and the conformational preferences of the polypeptide.

  • 3.
    Abelein, Axel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The zinc ion – a minimal chaperone mimicking agent forretardation of amyloid β peptide fibril formationManuscript (preprint) (Other academic)
  • 4.
    Abelein, Axel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Zinc as chaperone-mimicking agent for retardation of amyloid beta peptide fibril formation2015In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 112, no 17, p. 5407-5412Article in journal (Refereed)
    Abstract [en]

    Metal ions have emerged to play a key role in the aggregation process of amyloid beta (A beta) peptide that is closely related to the pathogenesis of Alzheimer's disease. A detailed understanding of the underlying mechanistic process of peptide-metal interactions, however, has been challenging to obtain. By applying a combination of NMR relaxation dispersion and fluorescence kinetics methods we have investigated quantitatively the thermodynamic A beta-Zn2+ binding features as well as how Zn2+ modulates the nucleation mechanism of the aggregation process. Our results show that, under near-physiological conditions, substoichiometric amounts of Zn2+ effectively retard the generation of amyloid fibrils. A global kinetic profile analysis reveals that in the absence of zinc A beta(40) aggregation is driven by a monomer-dependent secondary nucleation process in addition to fibril-end elongation. In the presence of Zn2+, the elongation rate is reduced, resulting in reduction of the aggregation rate, but not a complete inhibition of amyloid formation. We show that Zn2+ transiently binds to residues in the N terminus of the monomeric peptide. A thermodynamic analysis supports a model where the N terminus is folded around the Zn2+ ion, forming a marginally stable, short-lived folded A beta(40) species. This conformation is highly dynamic and only a few percent of the peptide molecules adopt this structure at any given time point. Our findings suggest that the folded A beta(40)-Zn2+ complex modulates the fibril ends, where elongation takes place, which efficiently retards fibril formation. In this conceptual framework we propose that zinc adopts the role of a minimal antiaggregation chaperone for A beta(40).

  • 5.
    Abelein, Axel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jarvet, Jüri
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. The National Institute of Chemical Physics and Biophysics, Estonia.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ionic Strength Modulation of the Free Energy Landscape of A beta(40) Peptide Fibril Formation2016In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 138, no 21, p. 6893-6902Article in journal (Refereed)
    Abstract [en]

    Protein misfolding and formation of cross-beta structured amyloid fibrils are linked to, many neurodegenerative disorders. Although recently developed,quantitative approaches have started to reveal the molecular nature of self-assembly and fibril formation of proteins and peptides, it is yet unclear how these self-organization events are precisely modulated by microenvironmental factors, which are known to strongly affect the macroscopic aggregation properties. Here, we characterize the explicit effect of ionic strength on the microscopic aggregation rates of amyloid beta peptide (A beta 40) self-association, implicated in Alzheimer's disease. We found that physiological ionic strength accelerates A beta 40 aggregation kinetics by promoting surface-catalyzed secondary nucleation reactions. This promoted catalytic effect can be assigned to shielding of electrostatic repulsion between Monomers on the fibril surface or between the fibril surface itself and monomeric peptides. Furthermore, we observe the formation of two different beta-structured states with =similar but distinct spectroscopic features, which can be assigned to an off-pathway immature state (F-beta*) and a mature stable State (F-beta), where salt favors formation of the F-beta fibril morphology. Addition of salt to preformed F-beta* accelerates transition to F-beta, underlining the dynamic nature of A beta 40 fibrils in solution. On the basis of,these results we suggest a model where salt decreases the free-energy barrier for A beta 40 folding to the F-beta state, favoring the buildup of the mature fibril morphology while omitting competing, energetically less favorable structural states.

  • 6.
    Abelein, Axel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kaspersen, Jørn Døvling
    Nielsen, Søren Bang
    Jensen, Grethe Vestergaard
    Christiansen, Gunna
    Pedersen, Jan Skov
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Otzen, Daniel E.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Formation of dynamic soluble surfactant-induced amyloid β peptide aggregation intermediates2013In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 32, p. 23518-23528Article in journal (Refereed)
    Abstract [en]

    Intermediate amyloidogenic states along the amyloid β peptide (Aβ) aggregation pathway have been shown to be linked to neurotoxicity. To shed more light on the different structures that may arise during Aβ aggregation, we here investigate surfactant-induced Aβ aggregation. This process leads to co-aggregates featuring a β-structure motif that is characteristic for mature amyloid-like structures. Surfactants induce secondary structure in Aβ in a concentration-dependent manner, from predominantly random coil at low surfactant concentration, via β-structure to the fully formed α-helical state at high surfactant concentration. The β-rich state is the most aggregation-prone as monitored by thioflavin T fluorescence. Small angle x-ray scattering reveals initial globular structures of surfactant-Aβ co-aggregated oligomers and formation of elongated fibrils during a slow aggregation process. Alongside this slow (minutes to hours time scale) fibrillation process, much faster dynamic exchange (k(ex) ∼1100 s(-1)) takes place between free and co-aggregate-bound peptide. The two hydrophobic segments of the peptide are directly involved in the chemical exchange and interact with the hydrophobic part of the co-aggregates. Our findings suggest a model for surfactant-induced aggregation where free peptide and surfactant initially co-aggregate to dynamic globular oligomers and eventually form elongated fibrils. When interacting with β-structure promoting substances, such as surfactants, Aβ is kinetically driven toward an aggregation-prone state.

  • 7.
    Abelein, Axel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lang, Lisa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lendel, Christofer
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Corrigendum to “Transient small molecule interactions kinetically modulate amyloid β peptide self-assembly” [FEBS Lett. 586 (2012) 3991–3995]2013In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 587, no 9, p. 1452-1452Article in journal (Other academic)
  • 8.
    Abelein, Axel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lang, Lisa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lendel, Christofer
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Transient small molecule interactions kinetically modulate amyloid beta peptide self-assembly2012In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 586, no 22, p. 3991-3995Article in journal (Refereed)
    Abstract [en]

    Small organic molecules, like Congo red and lacmoid, have been shown to modulate the self-assembly of the amyloid beta peptide (A beta). Here, we show that A beta forms NMR invisible non-toxic co-aggregates together with lacmoid as well as Congo red. We find that the interaction involves two distinct kinetic processes and at every given time point only a small fraction of A beta is in the co-aggregate. These weak transient interactions kinetically redirect the aggregation prone A beta from self-assembling into amyloid fibrils. These findings suggest that even such weak binders might be effective as therapeutics against pathogenic protein aggregation.

  • 9.
    Adrait, Annie
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Öhrström, Maria
    Barra, Anne-Laure
    The High Field Laboratory, CNRS/MPI, Grenoble, France.
    Thelander, Lars
    Department of Medical Biochemistry and Biophysicis, Umeå University.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    EPR studies on a stable sulfinyl radical observed in the iron-oxygen reconstituted Y177F/I263C protein double mutant of ribonucleotide reductase from mouse2002In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 41, no 20, p. 6510-6516Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductase (RNR) catalyzes the biosynthesis of deoxyribonucleotides. The active enzyme contains a diiron center and a tyrosyl free radical required for enzyme activity. The radical is located at Y177 in the R2 protein of mouse RNR. The radical is formed concomitantly with the μ-oxo-bridged diferric center in a reconstitution reaction between ferrous iron and molecular oxygen in the protein. EPR at 9.6 and 285 GHz was used to investigate the reconstitution reaction in the double-mutant Y177F/I263C of mouse protein R2. The aim was to produce a protein-linked radical derived from the Cys residue in the mutant protein to investigate its formation and characteristics. The mutation Y177F hinders normal radical formation at Y177, and the I263C mutation places a Cys residue at the same distance from the iron center as Y177 in the native protein. In the reconstitution reaction, we observed small amounts of a transient radical with a probable assignment to a peroxy radical, followed by a stable sulfinyl radical, most likely located on C263. The unusual radical stability may be explained by the hydrophobic surroundings of C263, which resemble the hydrophobic pocket surrounding Y177 in native protein R2. The observation of a sulfinyl radical in RNR strengthens the relationship between RNR and another free radical enzyme, pyruvate formate-lyase, where a similar relatively stable sulfinyl radical has been observed in a mutant. Sulfinyl radicals may possibly be considered as stabilized forms of very short-lived thiyl radicals, proposed to be important intermediates in the radical chemistry of RNR.

  • 10. Akishiba, Misao
    et al.
    Takeuchi, Toshihide
    Kawaguchi, Yoshimasa
    Sakamoto, Kentarou
    Yu, Hao-Hsin
    Nakase, Ikuhiko
    Takatani-Nakase, Tomoka
    Madani, Fatemeh
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Futaki, Shiroh
    Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide2017In: Nature Chemistry, ISSN 1755-4330, E-ISSN 1755-4349, Vol. 9, no 8, p. 751-761Article in journal (Refereed)
    Abstract [en]

    One of the major obstacles in intracellular targeting using antibodies is their limited release from endosomes into the cytosol. Here we report an approach to deliver proteins, which include antibodies, into cells by using endosomolytic peptides derived from the cationic and membrane-lytic spider venom peptide M-lycotoxin. The delivery peptides were developed by introducing one or two glutamic acid residues into the hydrophobic face. One peptide with the substitution of leucine by glutamic acid (L17E) was shown to enable a marked cytosolic liberation of antibodies (immunoglobulins G (IgGs)) from endosomes. The predominant membrane-perturbation mechanism of this peptide is the preferential disruption of negatively charged membranes (endosomal membranes) over neutral membranes (plasma membranes), and the endosomolytic peptide promotes the uptake by inducing macropinocytosis. The fidelity of this approach was confirmed through the intracellular delivery of a ribosome-inactivation protein (saporin), Cre recombinase and IgG delivery, which resulted in a specific labelling of the cytosolic proteins and subsequent suppression of the glucocorticoid receptor-mediated transcription. We also demonstrate the L17E-mediated cytosolic delivery of exosome-encapsulated proteins.

  • 11.
    Andersson, August
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    A kinetic model for peptide-induced leakage from vesicles and cells2007Conference paper (Other (popular science, discussion, etc.))
  • 12.
    Andersson, Charlotta S.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Öhrström, Maria
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Popović-Bijelić, Ana
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The manganese ion of the heterodinuclear Mn/Fe cofactor in Chlamydia trachomatis ribonucleotide reductase R2c is located at metal position 1.2012In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 134, no 1, p. 123-125Article in journal (Refereed)
    Abstract [en]

    The essential catalytic radical of Class-I ribonucleotide reductase is generated and delivered by protein R2, carrying a dinuclear metal cofactor. A new R2 subclass, R2c, prototyped by the Chlamydia trachomatis protein was recently discovered. This protein carries an oxygen-activating heterodinuclear Mn(II)/Fe(II) metal cofactor and generates a radical-equivalent Mn(IV)/Fe(III) oxidation state of the metal site, as opposed to the tyrosyl radical generated by other R2 subclasses. The metal arrangement of the heterodinuclear cofactor remains unknown. Is the metal positioning specific, and if so, where is which ion located? Here we use X-ray crystallography with anomalous scattering to show that the metal arrangement of this cofactor is specific with the manganese ion occupying metal position 1. This is the position proximal to the tyrosyl radical site in other R2 proteins and consistent with the assumption that the high-valent Mn(IV) species functions as a direct substitute for the tyrosyl radical.

  • 13.
    Assarsson, Maria
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Andersson, M E
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Persson, B O
    Sahlin, M
    Barra, A L
    Sjöberg, B M
    Nordlund, P
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Restoring proper radical generation by azide binding to the iron site of the E238A mutant R2 protein of ribonucleotide reductase from Escherichia coli.2001In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 29, p. 26852-26859Article in journal (Refereed)
    Abstract [en]

    The enzyme activity of Escherichia coli ribonucleotide reductase requires the presence of a stable tyrosyl free radical and diiron center in its smaller R2 component. The iron/radical site is formed in a reconstitution reaction between ferrous iron and molecular oxygen in the protein. The reaction is known to proceed via a paramagnetic intermediate X, formally a Fe(III)-Fe(IV) state. We have used 9.6 GHz and 285 GHz EPR to investigate intermediates in the reconstitution reaction in the iron ligand mutant R2 E238A with or without azide, formate, or acetate present. Paramagnetic intermediates, i.e. a long-living X-like intermediate and a transient tyrosyl radical, were observed only with azide and under none of the other conditions. A crystal structure of the mutant protein R2 E238A/Y122F with a diferrous iron site complexed with azide was determined. Azide was found to be a bridging ligand and the absent Glu-238 ligand was compensated for by azide and an extra coordination from Glu-204. A general scheme for the reconstitution reaction is presented based on EPR and structure results. This indicates that tyrosyl radical generation requires a specific ligand coordination with 4-coordinate Fe1 and 6-coordinate Fe2 after oxygen binding to the diferrous site.

  • 14. Bacher, Felix
    et al.
    Doemoetoer, Orsolya
    Kaltenbrunner, Maria
    Mojovic, Milos
    Popovic-Bijelic, Ana
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ozarowski, Andrew
    Filipovic, Lana
    Radulovic, Sinisa
    Enyedy, Eva A.
    Arion, Vladimir B.
    Effects of Terminal Dimethylation and Metal Coordination of Proline-2-formylpyridine Thiosemicarbazone Hybrids on Lipophilicity, Antiproliferative Activity, and hR2 RNR Inhibition2014In: Inorganic Chemistry, ISSN 0020-1669, E-ISSN 1520-510X, Vol. 53, no 23, p. 12595-12609Article in journal (Refereed)
    Abstract [en]

    The nickel(II), copper(II), and zinc(II) complexes of the proline-thiosemicarbazone hybrids 3-methyl-(S)-pyrrolidine-2-carboxylate-2-formylpyridine thiosemicarbazone (l-Pro-FTSC or (S)-H2L1) and 3-methyl-(R)-pyrrolidine-2-carboxylate-2-formylpyridine thiosemicarbazone (d-Pro-FTSC or (R)-H2L1), as well as 3-methyl-(S)-pyrrolidine-2-carboxylate-2-formylpyridine 4,4-dimethyl-thiosemicarbazone (dm-l-Pro-FTSC or (S)-H2L2), namely, [Ni(l-Pro-FTSC-2H)](2) (1), [Ni(d-Pro-FTSC-2H)](2) (2), [Ni(dm-l-Pro-FTSC-2H)](2) (3), [Cu(dm-l-Pro-FTSC-2H)] (6), [Zn(l-Pro-FTSC-2H)] (7), and [Zn(d-Pro-FTSC-2H)] (8), in addition to two previously reported, [Cu(l-Pro-FTSC-2H)] (4), [Cu(d-Pro-FTSC-2H)] (5), were synthesized and characterized by elemental analysis, one- and two-dimensional (1)H and (13)C NMR spectroscopy, circular dichroism, UV-vis, and electrospray ionization mass spectrometry. Compounds 1-3, 6, and 7 were also studied by single-crystal X-ray diffraction. Magnetic properties and solid-state high-field electron paramagnetic resonance spectra of 2 over the range of 50-420 GHz were investigated. The complex formation processes of l-Pro-FTSC with nickel(II) and zinc(II) were studied in aqueous solution due to the excellent water solubility of the complexes via pH potentiometry, UV-vis, and (1)H NMR spectroscopy. The results of the antiproliferative activity in vitro showed that dimethylation improves the cytotoxicity and hR2 RNR inhibition. Therefore, introduction of more lipophilic groups into thiosemicarbazone-proline backbone becomes an option for the synthesis of more efficient cytotoxic agents of this family of compounds.

  • 15. Björklund, Jörgen
    et al.
    Biverståhl, Henrik
    Gräslund, Astrid
    Mäler, Lena
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Real-time transmembrane translocation of penetratin driven by light-generated proton pumping.2006In: Biophys J, ISSN 0006-3495, Vol. 91, no 4, p. L29-31Article in journal (Refereed)
  • 16.
    Björnerås, Johannes
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Membrane Interaction of Disease-Related Dynorphin A Variants2013In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, no 24, p. 4157-4167Article in journal (Refereed)
    Abstract [en]

    The membrane interaction properties of two single-residue variants, R6W and L5S, of the 17-amino acid neuropeptide dynorphin A (DynA) were studied by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Corresponding gene mutations have recently been discovered in humans and causatively linked to a neurodegenerative disorder. The peptides were investigated in buffer and in isotropic solutions of q = 0.3 bicelles with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or DMPC (0.8) and 1,2-dimyristoyl-sn-glycero-3-phospho(1'-rac-glycerol) (DMPG) (0.2). The CD results and the NMR secondary chemical shifts show that R6W-DynA has a small a-helical fraction in buffer, which increases in the presence of bicelles, while L5S-DynA is mainly unstructured under all conditions studied here. R6W-DynA has an almost complete association with zwitterionic bicelles (similar to 90%, as probed by NMR diffusion experiments), similar to the behavior of wtDynA, while L5S-DynA has a weaker association (similar to 50%). For all peptides, the level of bicelle association is increased in negatively charged bicelles. The L5A-DynA peptide adopts a very shallow position in the headgroup region of the bicelle bilayer, as studied by paramagnetic spin relaxation enhancement experiments using paramagnetic probes. Similarly, the results show that R6W-DynA is more deeply buried in the bilayer, with only the C-terminal residues exposed to solvent, again more similar to the case of wild-type DynA. We suggest that the results presented here may explain the differences in cell toxicity of these disease-related neuropeptide variants.

  • 17.
    Björnerås, Johannes
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The membrane interaction of dynorphin A depends on lipid head-group chargeManuscript (preprint) (Other academic)
    Abstract [en]

    The influence of lipid bicelles on the dynamics of the opioid peptide DynA has been investigated by Nuclear Magnetic Resonance. DynA exerts its opioid effects mainly through interactions with the κ subtype of the opioid receptors, but has also been demonstrated to have direct interactions with membranes. Among other properties, it has been shown that the peptide causes membrane disruption and may penetrate bilayers. Despite the fact that DynA appears to bind tightly to model lipid bilayers, no structure induction has been observed. To further study the effect of membrane interactions we have here therefore measured the fast local dynamics of DynA specifically labeled with 15N in three backbone amide sites (Gly2, Leu5 and Leu12) in fast-tumbling bicelles, both with and without the incorporation of the negatively charged dimyristoylglycerol. We also examined the amide exchange in the two bicelles. We find that despite the fact that DynA is largely unstructured in both types of bicelles, the peptide has restricted backbone dynamics, which depends on the presence of negatively charged lipids. Moreover we see that the lipid dependence is not uniform throughout the sequence, but is most noticeable for Leu5, which precedes an unusually basic stretch of amino acid residues. The findings indicate that this basic sequence may be of significance for bilayer recognition. Finally, we note that the dynamical behavior of the peptide is much more influenced by the lipid surroundings than what the structural properties are.

  • 18.
    Björnerås, Johannes
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kurnik, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Direct detection of neuropeptide dynorphin A binding to the second extracellular loop of the kappa opioid receptor using a soluble protein scaffold2014In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 281, no 3, p. 814-824Article in journal (Refereed)
    Abstract [en]

    The molecular determinants for selectivity of ligand binding to membrane receptors are of key importance for the understanding of cellular signalling, as well as for rational therapeutic intervention. In the present study, we target the interaction between the kappa opioid receptor (KOR) and its native peptide ligand dynorphin A (DynA) using solution state NMR spectroscopy, which is generally made difficult by the sheer size of membrane bound receptors. Our method is based on 'transplantation' of an extracellular loop of KOR into a 'surrogate' scaffold; in this case, a soluble beta-barrel. Our results corroborate the general feasibility of the method, showing that the inserted receptor segment has negligible effects on the properties of the scaffold protein, at the same time as maintaining an ability to bind its native DynA ligand. Upon DynA binding, only small induced chemical shift changes of the KOR loop were observed, whereas chemical shift changes of DynA and NMR paramagnetic relaxation data show conclusively that the peptide interacts with the inserted loop. The binding interface is composed of a disordered part of the KOR loop and involves both electrostatic and hydrophobic interactions. Even so, simultaneous effects along the DynA sequence upon binding show that control of the recognition is a concerted event.

  • 19.
    Bárány-Wallje, Elsa
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Andersson, August
    Gräslund, Astrid
    Mäler, Lena
    Dynamics of transportan in bicelles is surface charge dependent.2006In: J Biomol NMR, ISSN 0925-2738, Vol. 35, no 2, p. 137-47Article in journal (Refereed)
    Abstract [en]

    In this study we investigated the dynamic behavior of the chimeric cell-penetrating peptide transportan in membrane-like environments using NMR. Backbone amide 15N spin relaxation was used to investigate the dynamics in two bicelles: neutral DMPC bicelles and partly negatively charged DMPG-containing bicelles.

    The structure of the peptide as judged from CD and chemical shifts is similar in the two cases. Both the overall motion as well as the local dynamics is, however, different in the two types of bicelles. The overall dynamics of the peptide is significantly slower in the partly negatively charged bicelle environment, as evidenced by longer global correlation times for all measured sites.

    The local motion, as judged from generalized order parameters, is for all sites in the peptide more restricted when bound to negatively charged bicelles than when bound to neutral bicelles (increase in S2 is on average 0.11±0.07). The slower dynamics of transportan in charged membrane model systems cause significant line broadening in the proton NMR spectrum, which in certain cases limits the observation of 1H signals for transportan when bound to the membrane. The effect of transportan on DMPC and DHPC motion in zwitterionic bicelles was also investigated, and the motion of both components in the bicelle was found to be affected.

  • 20.
    Bárány-Wallje, Elsa
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. biofysik.
    Andersson, August
    Gräslund, Astrid
    Mäler, Lena
    NMR solution structure and position of transportan in neutral phospholipid bicelles.2004In: FEBS Lett, ISSN 0014-5793, Vol. 567, no 2-3, p. 265-9Article in journal (Refereed)
    Abstract [en]

    Transportan is a chimeric cell-penetrating peptide constructed from the peptides galanin and mastoparan, which has the ability to internalize living cells carrying a hydrophilic load. In this study, we have determined the NMR solution structure and investigated the position of transportan in neutral bicelles. The structure revealed a well-defined -helix in the C-terminal mastoparan part of the peptide and a weaker tendency to form an -helix in the N-terminal domain. The position of the peptide in relation to the membrane, as studied by adding paramagnetic probes, shows that the peptide lies parallel to, and in the head-group region of the membrane surface. This result is supported by amide proton secondary chemical shifts.

  • 21.
    Bárány-Wallje, Elsa
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gaur, Jugnu
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lundberg, Pontus
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Differential membrane perturbation caused by the cell penetrating peptide Tp10 depending on attached cargo2007In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 581, no 13, p. 2389-2393Article in journal (Refereed)
    Abstract [en]

    The membrane leakage caused by the cell penetrating peptide Tp10, a variant of transportan, was studied in large unilamellar vesicles with the entrapped fluorophore calcein. The vesicles were composed of zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. A significant decrease in membrane leakage was found when the 55 kDa streptavidin protein was attached to Tp10. When a 5.4 kDa peptide nucleic acid molecule was attached, the membrane leakage was comparable to that caused by Tp10 alone. The results suggest that direct membrane effects may cause membrane translocation of Tp10 alone and of smaller complexes, whereas these effects do not contribute for larger cargoes.

  • 22.
    Bárány-Wallje, Elsa
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structure and dynamics of galanin in phospholipid bicelles2007Conference paper (Other (popular science, discussion, etc.))
  • 23. Chemerovski-Glikman, Marina
    et al.
    Rozentur-Shkop, Eva
    Richman, Michal
    Grupi, Asaf
    Getler, Asaf
    Cohen, Haim Y.
    Shaked, Hadassa
    Wallin, Cecilia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wärmländer, Sebastian K. T. S.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Haas, Elisha
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Chill, Jordan H.
    Rahimipour, Shai
    Self-Assembled Cyclic D,L-alpha-Peptides as Generic Conformational Inhibitors of the alpha-Synuclein Aggregation and Toxicity: In Vitro and Mechanistic Studies2016In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 22, no 40, p. 14236-14246Article in journal (Refereed)
    Abstract [en]

    Many peptides and proteins with large sequences and structural differences self-assemble into disease-causing amyloids that share very similar biochemical and biophysical characteristics, which may contribute to their cross-interaction. Here, we demonstrate how the self-assembled, cyclic D,L-alpha-peptide CP-2, which has similar structural and functional properties to those of amyloids, acts as a generic inhibitor of the Parkinson's disease associated alpha-synuclein (alpha-syn) aggregation to toxic oligomers by an, off-pathway mechanism. We show that CP-2 interacts with the N-terminal and the non-amyloid-beta component region of alpha-syn, which are responsible for alpha-syn's membrane intercalation and self-assembly, thus changing the overall conformation of alpha-syn. CP-2 also remodels alpha-syn fibrils to nontoxic amorphous species and permeates cells through endosomes/lysosomes to reduce the accumulation and toxicity of intracellular alpha-syn in neuronal cells overexpressing alpha-syn. Our studies suggest that targeting the common structural conformation of amyloids may be a promising approach for developing new therapeutics for amyloidogenic diseases.

  • 24.
    Danielsson, J
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Andersson, A
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jarvet, J
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, A
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    15N relaxation study of the amyloid beta-peptide structural propensities and persistence length.2006In: Magn Reson Chem, ISSN 0749-1581, Vol. 44, no S1, p. S114-21Article in journal (Refereed)
    Abstract [en]

    The dynamics of monomeric Alzheimer A(1-40) in aqueous solution was studied using heteronuclear NMR experiments. 15N NMR relaxation rates of amide groups report on the dynamics in the peptide chain and make it possible to estimate structural propensities from temperature-dependent relaxation data and chemical shifts change analysis. The persistence length of the polypeptide chain was determined using a model in which the influence of neighboring residue relaxation is assumed to decay exponentially as a function of distance. The persistence length of the A(1-40) monomer was found to decrease from eight to three residues when temperature was increased from 3 to 18 °C. At 3 °C the peptide shows structural propensities that correlate well with the suggested secondary structure regions of the peptide to be present in the fibrils, and with the -helical structure in membrane-mimicking systems. Our data leads to a structural model for the monomeric soluble -peptide with six different regions of secondary structure propensities. The peptide has two regions with -strand propensity (residues 16-24 and 31-40), two regions with high PII-helix propensity (residues 1-4 and 11-15) and two unstructured regions with higher mobility (residues 5-10 and 25-30) connecting the structural elements. Copyright © 2006 John Wiley & Sons, Ltd.

  • 25.
    Danielsson, Jens
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pierattelli, Roberta
    Banci, Lucia
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    High-resolution NMR studies of the sinc-binding site of the Alzheimer’s Aβ-peptide2007In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 274, no 1, p. 46-59Article in journal (Refereed)
  • 26.
    Davydov, Albert
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Öhrström, Maria
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Liu, Aimin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Thelander, Lars
    Department of Medical Biosciences, Medical Biochemistry, Umeå Universtity.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Chemical reduction of the diferric/radical center in protein R2 from mouse ribonucleotide reductase is independent of the proposed radical transfer pathway2002In: Inorganica Chimica Acta, ISSN 0020-1693, E-ISSN 1873-3255, Vol. 331, no 1, p. 65-72Article in journal (Refereed)
    Abstract [en]

    The rates of reduction of the diferric/radical center in mouse ribonucleotide reductase protein R2 were studied by light absorption and EPR in the native protein and in three point mutants of conserved residues involved in the proposed radical transfer pathway (D266A, W103Y) or in the unstructured C terminal domain (Y370W). The pseudo-first order rate constants for chemical reduction of the tyrosyl radical and diferric center by hydroxyurea, sodium dithionite or the dihydro form of flavin adenine dinucleotide, were comparable with or higher (particularly D266A, by dithionite) than in native R2. Molecular modeling of the D266A mutant showed that the iron/radical site should be more accessible for external reductants in the mutant than in native R2. The results indicate that no specific pathway is required for the reduction. The dihydro form of flavin adenine dinucleotide was found to be a very efficient reductant in the studied proteins compared to dithionite alone. The EPR spectra of the mixed-valent Fe(II)Fe(III) sites formed by chemical reduction in the D266A and W103Y mutants were clearly different from the spectrum observed in the native protein, indicating that the structure of the diferric site was affected by the mutations, as also suggested by the modeling study. No difference was observed between the mixed-valent EPR spectra generated by chemical reduction in Y370W mutant and native mouse R2 protein

  • 27. Dobrov, Anatolie
    et al.
    Goeschl, Simone
    Jakupec, Michael A.
    Popovic-Bijelic, Ana
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rapta, Peter
    Arion, Vladimir B.
    A highly cytotoxic modified paullone ligand bearing a TEMPO free-radical unit and its copper(II) complex as potential hR2 RNR inhibitors2013In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 49, no 85, p. 10007-10009Article in journal (Refereed)
    Abstract [en]

    A new paullone-TEMPO conjugate and its copper(II) complex inhibit RNR activity and show high antiproliferative activity in human cancer cell lines.

  • 28. Esbjörner, Elin K.
    et al.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nordén, Bengt
    Membrane Interactions of Cell-Penetrating Peptides2007In: Handbook of Cell-Penetrating Peptides / [ed] Ülo Langel, Boca Raton: CRC Press, 2007, 2, p. 109-137Chapter in book (Refereed)
  • 29.
    Esbjörner, Elin K.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oglęcka, Kamila
    Lincoln, Per
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nordén, Bengt
    Membrane binding of pH-sensitive Influenza fusion peptides. Positioning, configuration and induced leakage in lipid vesicles models2007In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 47, p. 13490-13504Article in journal (Refereed)
    Abstract [en]

    pH-sensitive HA2 fusion peptides from influenza virus hemagglutinin have potential as endosomal escape-inducing components in peptide-based drug delivery. Polarized light spectroscopy and tryptophan fluorescence were used to assess the conformation, orientation, effect on lipid order, and binding kinetics of wild-type peptide HA 1-23) and a glutamic acid-enriched analogue (INF7) in large unilamellar POPC or POPC/POPG (4:1) lipid vesicles (LUVs). pH-sensitive membrane leakage was established for INF7 but not HA2(1-23) using an entrapped-dye assay. A correlation is indicated between leakage and a low degree of lipid chain order (assessed by linear dichroism, LD, of the membrane orientation probe retinoic acid). Both peptides display poor alignment in zwitterionic POPC LUVs compared to POPC/POPG (4:1) LUVs, and it was found that peptide-lipid interactions display slow kinetics (hours), resulting in reduced lipid order and increased tryptophan shielding. At pH 7.4, INF7 displays tryptophan emission and LD features indicative of a surface-orientated peptide, suggesting that its N-terminal glutamic acid residues prevent deep penetration into the hydrocarbon core. At pH 5.0, INF7 displays weaker LD signals, indicating poor orientation, possibly due to aggregation. By contrast, the orientation of the HA2(1-23) peptide backbone supports previously reported oblique insertion (∼60-65° relative to the membrane normal), and aromatic side-chain orientations are consistent with an interfacial (pH-independent) location of the C-terminus. We propose that a conformational change upon reduction of pH is limited to minor rearrangements of the peptide "hinge region" around Trp14 and repositioning of this residue

  • 30.
    Georgieva, Elka R.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Narvaez, Ana Julia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hedin, Niklas
    Stockholm University, Faculty of Science, Department of Physical, Inorganic and Structural Chemistry.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Secondary structure conversions of Mycobacterium tuberculosis ribonucleotide reductase protein R2 under varying pH and temperature conditions2008In: Biophysical Chemistry, Vol. 137, no 43-48Article in journal (Refereed)
    Abstract [en]

    The structural properties of Mycobacterium tuberculosis (Mtb) ribonucleotide reductase R2 protein were studied under varying pH and temperature conditions by circular dichroism (CD) spectroscopy as well as dynamic light scattering (DLS). Under physiological conditions this protein has a high alpha-helical content, similar to the corresponding protein from other species, e.g. mouse. Decreasing the pH induced significant structure conversions. When pH was below 6.5 an aggregated structure was observed and reached a maximum at pH 4. The aggregated state of this protein was verified by DLS and was found to be rich in beta-structure. This amyloid-like structure transformed into a molten globule state with high temperature stability (between 25 and 80 degrees C) at pH below 3. The corresponding mouse protein R2 under similar conditions showed no evidence of an aggregated state around pH 4.

  • 31. Ghalebani, Leila
    et al.
    Wahlström, Anna
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wärmländer, Sebastian
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    pH dependence of the specific binding of Cu(II) and Zn(II) ions to the amyloid β peptideManuscript (preprint) (Other academic)
  • 32.
    Ghalebani, Leila
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wahlström, Anna
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wärmländer, Sebastian
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    pH-dependence of the specific binding of Cu(II) and Zn(II) ions to the amyloid-beta peptide2012In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 421, no 3, p. 554-560Article in journal (Refereed)
    Abstract [en]

    Metal ions like Cu(II) and Zn(II) are accumulated in Alzheimer's disease amyloid plaques. The amyloid-beta (A beta) peptide involved in the disease interacts with these metal ions at neutral pH via ligands provided by the N-terminal histidines and the N-terminus. The present study uses high-resolution NMR spectroscopy to monitor the residue-specific interactions of Cu(II) and Zn(II) with N-15- and C-13,N-15-labeled A beta(1-40) peptides at varying pH levels. At pH 7.4 both ions bind to the specific ligands, competing with one another. At pH 5.5 Cu(II) retains its specific histidine ligands, while Zn(II) seems to lack residue-specific interactions. The low pH mimics acidosis which is linked to inflammatory processes in vivo. The results suggest that the cell toxic effects of redox active Cu(II) binding to AD may be reversed by the protective activity of non-redox active Zn(II) binding to the same major binding site under non-acidic conditions. Under acidic conditions, the protective effect of Zn(II) may be decreased or changed, since Zn(II) is less able to compete with Cu(II) for the specific binding site on the AD peptide under these conditions.

  • 33.
    Griese, Julia J.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Roos, Katarina
    Stockholm University, Faculty of Science, Department of Physics.
    Cox, Nicholas
    Shafaat, Hannah S.
    Branca, Rui M. M.
    Lehtio, Janne
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lubitz, Wolfgang
    Siegbahn, Per E. M.
    Stockholm University, Faculty of Science, Department of Physics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Direct observation of structurally encoded metal discrimination and ether bond formation in a heterodinuclear metalloprotein2013In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 110, no 43, p. 17189-17194Article in journal (Refereed)
    Abstract [en]

    Although metallocofactors are ubiquitous in enzyme catalysis, how metal binding specificity arises remains poorly understood, especially in the case of metals with similar primary ligand preferences such as manganese and iron. The biochemical selection of manganese over iron presents a particularly intricate problem because manganese is generally present in cells at a lower concentration than iron, while also having a lower predicted complex stability according to the Irving-Williams series (Mn-II < Fe-II < Ni-II < Co-II < Cu-II > Zn-II). Here we show that a heterodinuclear Mn/Fe cofactor with the same primary protein ligands in both metal sites self-assembles from MnII and FeII in vitro, thus diverging from the Irving-Williams series without requiring auxiliary factors such as metallochaperones. Crystallographic, spectroscopic, and computational data demonstrate that one of the two metal sites preferentially binds FeII over MnII as expected, whereas the other site is nonspecific, binding equal amounts of both metals in the absence of oxygen. Oxygen exposure results in further accumulation of the Mn/Fe cofactor, indicating that cofactor assembly is at least a two-step process governed by both the intrinsic metal specificity of the protein scaffold and additional effects exerted during oxygen binding or activation. We further show that the mixed-metal cofactor catalyzes a two-electron oxidation of the protein scaffold, yielding a tyrosine-valine ether cross-link. Theoretical modeling of the reaction by density functional theory suggests a multistep mechanism including a valyl radical intermediate.

  • 34.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Biophysical studies of model membrane interactions of cell penetrating peptides2007Conference paper (Other (popular science, discussion, etc.))
  • 35.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ribonucleotide reductase - a free radical enzyme2007Conference paper (Other (popular science, discussion, etc.))
  • 36.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ribonucleotide reductase - EPR studies of free radicals and redox-active metal sites2007Conference paper (Other (popular science, discussion, etc.))
  • 37.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ribonucleotide reductase - interplay between redox-active metals and free radicals2007Conference paper (Other (popular science, discussion, etc.))
  • 38.
    Gräslund, Astrid
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pierattelli, R.
    Banci, L.
    Metal ion interaction with the Alzheimer A-beta peptide2007Conference paper (Other (popular science, discussion, etc.))
  • 39.
    Gräslund, Astrid
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ehrenberg, Anders
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Tyrosyl radical, diiron center and enzyme mechanism in ribonucleotide reductase2007In: Appl. Magn. Reson., no 31, p. 447-455Article in journal (Refereed)
  • 40.
    Gräslund, Astrid
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Testing membrane interactions of CPPs2011In: Methods in molecular biology (Clifton, N.J.), ISSN 1940-6029, Vol. 683, p. 33-40Article in journal (Refereed)
    Abstract [en]

    The chapter deals with some biophysical methods used for investigating CPP-induced changes in membrane properties by spectroscopy methods such as fluorescence or NMR and methods used for probing CPP-induced leakage in membranes. Some useful model systems for biomembranes are described. These include large unilamellar phospholipid vesicles (LUVs) of well-defined size (diameter typically 100 nm). A protocol for the preparation of such vesicles is included. The leakage studies make use of LUVs with entrapped dye molecules. The NMR studies make use of mixed micelles (bicelles) as a membrane mimetic system, which can be oriented in the magnetic field of the spectrometer.

  • 41.
    Guterstam, Peter
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Madani, Fatemeh
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hirose, Hisaaki
    Takeuchi, Toshihide
    Futaki, Shiroh
    EL Andaloussi, Samir
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Elucidating cell-penetrating peptide mechanisms of action for membrane interaction, cellular uptake, and translocation utilizing the hydrophobic counter-anion pyrenebutyrate2009In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1788, no 12, p. 2509-2517Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are membrane permeable vectors recognized for their intrinsic ability to gain access to the cell interior. The hydrophobic counter-anion, pyrenebutyrate, enhances cellular uptake of oligoarginine CPPs. To elucidate CPP uptake mechanisms, the effect of pyrenebutyrate on well-recognized CPPs with various hydrophobicity and arginine content is investigated. The cellular CPP-uptake and CPP-mediated oligonucleotide delivery is analyzed by fluorescence activated cell sorting, confocal microscopy, and a cell based splice-switching assay. The splice-switching oligonucleotide is a mixmer of 2’-O-methyl RNA and locked nucleic acids delivered as a non-covalent complex with 10-fold molar CPP excess. CPP-induced membrane perturbation on large unilamellar vesicles is investigated in calcein release experiments. We observed that pyrenebutyrate facilitates cellular uptake and translocation of oligonucleotide mediated by oligoarginine nonamer while limited effect of pyrenebutyrate on more hydrophobic CPPs was observed. By combining the different experimental results we conclude that the pathway for cellular uptake of oligoarginine is dominated by direct membrane translocation, whereas the pathway for oligoarginine-mediated oligonucleotide translocation is dominated by endocytosis. Both mechanisms are promoted by pyrenebutyrate and we suggest that pyrenebutyrate has different sites of action for the two uptake and translocation mechanisms.

  • 42.
    Hammerstad, Marta
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. University of Oslo, Norway.
    Rohr, Asmund K.
    Andersen, Niels H.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Andersson, K. Kristoffer
    The class Ib ribonucleotide reductase from Mycobacterium tuberculosis has two active R2F subunits2014In: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 19, no 6, p. 893-902Article in journal (Refereed)
    Abstract [en]

    Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to their corresponding deoxyribonucleotides, playing a crucial role in DNA repair and replication in all living organisms. Class Ib RNRs require either a diiron-tyrosyl radical (Y center dot) or a dimanganese-Y center dot cofactor in their R2F subunit to initiate ribonucleotide reduction in the R1 subunit. Mycobacterium tuberculosis, the causative agent of tuberculosis, contains two genes, nrdF1 and nrdF2, encoding the small subunits R2F-1 and R2F-2, respectively, where the latter has been thought to serve as the only active small subunit in the M. tuberculosis class Ib RNR. Here, we present evidence for the presence of an active Fe (2) (III) -Y center dot cofactor in the M. tuberculosis RNR R2F-1 small subunit, supported and characterized by UV-vis, X-band electron paramagnetic resonance, and resonance Raman spectroscopy, showing features similar to those for the M. tuberculosis R2F-2-Fe (2) (III) -Y center dot cofactor. We also report enzymatic activity of Fe (2) (III) -R2F-1 when assayed with R1, and suggest that the active M. tuberculosis class Ib RNR can use two different small subunits, R2F-1 and R2F-2, with similar activity.

  • 43. Heffeter, P.
    et al.
    Popovic-Bijelic, Ana
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Saiko, P.
    Dornetshuber, R.
    Jungwirth, U.
    Voevodskaya, Nina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Biglino, D.
    Jakupec, M. A.
    Elbling, L.
    Micksche, M.
    Szekeres, T.
    Keppler, B. K.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Berger, W.
    Ribonucleotide Reductase as One Important Target of [Tris(1,10- phenanthroline)lanthanum(III)] Trithiocyanate (KP772)2009In: Current Cancer Drug Targets, ISSN 1568-0096, E-ISSN 1873-5576, Vol. 9, no 5, p. 595-607Article in journal (Refereed)
    Abstract [en]

    KP772 is a new lanthanum complex containing three 1,10-phenathroline molecules. Recently, we have demonstrated that the promising in vitro and in vivo anticancer properties of KP772 are based on p53-independent G(0)G(1) arrest and apoptosis induction. A National Cancer Institute (NCI) screen revealed significant correlation of KP772 activity with that of the ribonucleotide reductase (RR) inhibitor hydroxyurea (HU). Consequently, this study aimed to investigate whether KP772 targets DNA synthesis in tumor cells by RR inhibition. Indeed, KP772 treatment led to significant reduction of cytidine incorporation paralleled by a decrease of deoxynucleoside triphosphate (dNTP) pools. This strongly indicates disruption of RR activity. Moreover, KP772 protected against oxidative stress, suggesting that this drug might interfere with RR by interaction with the tyrosyl radical in subunit R2. Additionally, several observations (e.g. increase of transferrin receptor expression and protective effect of iron preloading) indicate that KP772 interferes with cellular iron homeostasis. Accordingly, co-incubation of Fe(II) with KP772 led to generation of a coloured iron complex (Fe-KP772) in cell free systems. In electron paramagnetic resonance (EPR) measurements of mouse R2 subunits, KP772 disrupted the tyrosyl radical while Fe-KP772 had no significant effects. Moreover, coincubation of KP772 with iron-loaded R2 led to formation of Fe-KP772 suggesting chelation of RR-bound Fe(II). Summarizing, our data prove that KP772 inhibits RR by targeting the iron centre of the R2 subunit. As also Fe-KP772 as well as free lanthanum exert significant -though less pronounced- cytotoxic/static activities, additional mechanisms are likely to synergise with RR inhibition in the promising anticancer activity of KP772.

  • 44. Horvath, Istvan
    et al.
    Iashchishyn, Igor A.
    Moskalenko, Roman A.
    Wang, Chao
    Wärmländer, Sebastian K. T. S.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wallin, Cecilia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kovacs, Gabor G.
    Morozova-Roche, Ludmilla A.
    Co-aggregation of pro-inflammatory S100A9 with alpha-synuclein in Parkinson's disease: ex vivo and in vitro studies2018In: Journal of Neuroinflammation, ISSN 1742-2094, E-ISSN 1742-2094, Vol. 15, article id 172Article in journal (Refereed)
    Abstract [en]

    Background: Chronic neuroinflammation is a hallmark of Parkinson's disease (PD) pathophysiology, associated with increased levels of pro-inflammatory factors in PD brain tissues. The pro-inflammatory mediator and highly amyloidogenic protein S100A9 is involved in the amyloid-neuroinflammatory cascade in Alzheimer's disease. This is the first report on the co-aggregation of alpha-synuclein (alpha-syn) and S100A9 both in vitro and ex vivo in PD brain. Methods: Single and sequential immunohistochemistry, immunofluorescence, scanning electron and atomic force (AFM) microscopies were used to analyze the ex vivo PD brain tissues for S100A9 and alpha-syn location and aggregation. In vitro studies revealing S100A9 and alpha-syn interaction and co-aggregation were conducted by NMR, circular dichroism, Thioflavin-T fluorescence, AFM, and surface plasmon resonance methods. Results: Co-localized and co-aggregated S100A9 and alpha-syn were found in 20% Lewy bodies and 77% neuronal cells in the substantia nigra; both proteins were also observed in Lewy bodies in PD frontal lobe (Braak stages 4-6). Lewy bodies were characterized by ca. 10-23 mu m outer diameter, with S100A9 and alpha-syn being co-localized in the same lamellar structures. S100A9 was also detected in neurons and blood vessels of the aged patients without PD, but in much lesser extent. In vitro S100A9 and alpha-syn were shown to interact with each other via the alpha-syn C-terminus with an apparent dissociation constant of ca. 5 mu M. Their co-aggregation occurred significantly faster and led to formation of larger amyloid aggregates than the self-assembly of individual proteins. S100A9 amyloid oligomers were more toxic than those of alpha-syn, while co-aggregation of both proteins mitigated the cytotoxicity of S100A9 oligomers. Conclusions: We suggest that sustained neuroinflammation promoting the spread of amyloidogenic S100A9 in the brain tissues may trigger the amyloid cascade involving alpha-syn and S100A9 and leading to PD, similar to the effect of S100A9 and A beta co-aggregation in Alzheimer's disease. The finding of S100A9 involvement in PD may open a new avenue for therapeutic interventions targeting S100A9 and preventing its amyloid self-assembly in affected brain tissues.

  • 45.
    Hugonin, Loïc
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Perálvarez-Marín, Alex
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Secondary structure transitions and aggregation induced in dynorphin neuropeptides by the detergent sodium dodecyl sulfate.2008In: Biochim Biophys Acta, ISSN 0006-3002, Vol. 1778, no 11, p. 2580-7Article in journal (Refereed)
    Abstract [en]

    Dynorphins, endogeneous opioid neuropeptides, function as ligands to the opioid kappa receptors and also induce non-opioid effects in neurons, probably related to direct membrane interactions. We have characterized the structure transitions of dynorphins (big dynorphin, dynorphin A and dynorphin B) induced by the detergent sodium dodecyl sulfate (SDS). In SDS titrations monitored by circular dichroism, we observed secondary structure conversions of the peptides from random coil to alpha-helix with a highly aggregated intermediate. As determined by Fourier transform infrared spectroscopy, this intermediate exhibited beta-sheet structure for dynorphin B and big dynorphin. In contrast, aggregated dynorphin A was alpha-helical without considerable beta-sheet content. Hydrophobicity analysis indicates that the YGGFLRR motif present in all dynorphins is prone to be inserted in the membrane. Comparing big dynorphin with dynorphin A and dynorphin B, we suggest that the potent neurotoxicity of big dynorphin could be related to the combination of amino acid sequences and secondary structure propensities of dynorphin A and dynorphin B, which may generate a synergistic effect for big dynorphin membrane perturbing properties. The induced aggregated alpha-helix of dynorphin A is also correlated with membrane perturbations, whereas the beta-sheet of dynorphin B does not correlate with membrane perturbations.

  • 46.
    Kilk, Kalle
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology. University of Tartu, Estonia.
    Magzoub, Mazin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pooga, Margus
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology. Estonian Biocenter, Estonia.
    Eriksson, L. E. Göran
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry and Neurotoxicology.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Cellular internalization of a cargo complex with a novel peptide derived from the third helix of the islet-1 homeodomain: Comparison with the penetratin peptide2001In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 12, no 6, p. 911-916Article in journal (Refereed)
    Abstract [en]

    Cellular translocation into a human Bowes melanoma cell line was investigated and compared for penetratin and pIsl, two peptides that correspond to the third helices of the related homeodomains, from the Antennapedia transcription factor of Drosophila and the rat insulin-1 gene enhancer protein, respectively. Both biotinylated peptides internalized into the cells with similar efficacy, yielding an analogous intracellular distribution. When a large cargo protein, 63 kDa avidin, was coupled to either peptide, efficient cellular uptake for both the peptide−protein complexes was observed. The interactions between each peptide and SDS micelles were studied by fluorescence spectroscopy and acrylamide quenching of the intrinsic tryptophan (Trp) fluorescence. Both peptides interacted strongly and almost identically with the membrane mimicking environment. Compared to penetratin, the new transport peptide pIsl has only one Trp residue, which simplifies the interpretation of the fluorescence spectra and in addition has a native Cys residue, which may be used for alternative coupling reactions of cargoes of different character.

  • 47. Kolberg, Matthias
    et al.
    Bleifuss, Günter
    Sjöberg, Britt-Marie
    Stockholm University.
    Gräslund, Astrid
    Stockholm University.
    Lubitz, Wolfgang
    Lendzian, Friedhelm
    Lassmann, Günter
    Generation and Electron Paramagnetic Resonance Spin Trapping Detection of Thinyl Radicals in Model Proteins and in the R1 Subunit of Escherichia coli2002In: Archives of Biochemistry & Biophysics, Vol. 397, p. 57-68Article in journal (Refereed)
  • 48. Kolberg, Matthias
    et al.
    Logan, Derek
    Bleifuss, Günther
    Pötsch, Stephan
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Molecular Biology and Functional Genomics.
    Lubitz, Wolfgang
    Lassmann, Günter
    Lendzian, Friedhelm
    A new tyrosyl radical on Phe208 as ligand to the diiron center in Escherichia coli ribonucleotide reductase, mutant R2-Y122H. Combined x-ray diffraction and EPR/ENDOR studies.2005In: J Biol Chem, ISSN 0021-9258, Vol. 280, no 12, p. 11233-46Article in journal (Other academic)
    Abstract [en]

    The R2 protein subunit of class I ribonucleotide reductase (RNR) belongs to a structurally related family of oxygen bridged diiron proteins. In wild-type R2 of Escherichia coli, reductive cleavage of molecular oxygen by the diferrous iron center generates a radical on a nearby tyrosine residue (Tyr122), which is essential for the enzymatic activity of RNR, converting ribonucleotides into deoxyribonucleotides. In this work, we characterize the mutant E. coli protein R2-Y122H, where the radical site is substituted with a histidine residue. The x-ray structure verifies the mutation. R2-Y122H contains a novel stable paramagnetic center which we name H, and which we have previously proposed to be a diferric iron center with a strongly coupled radical, Fe(III)Fe(III)R.. Here we report a detailed characterization of center H, using 1H/2H -14N/15N- and 57Fe-ENDOR in comparison with the Fe(III)Fe(IV) intermediate X observed in the iron reconstitution reaction of R2. Specific deuterium labeling of phenylalanine residues reveals that the radical results from a phenylalanine. As Phe208 is the only phenylalanine in the ligand sphere of the iron site, and generation of a phenyl radical requires a very high oxidation potential, we propose that in Y122H residue Phe208 is hydroxylated, as observed earlier in another mutant (R2-Y122F/E238A), and further oxidized to a phenoxyl radical, which is coordinated to Fe1. This work demonstrates that small structural changes can redirect the reactivity of the diiron site, leading to oxygenation of a hydrocarbon, as observed in the structurally similar methane monoxygenase, and beyond, to formation of a stable iron-coordinated radical.

  • 49. Kolberg, Matthis
    et al.
    Bleifuss, Günter
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sjöberg, Britt-Marie
    Department of Molecular Biology and Functional Genomics.
    Lubitz, Wolfgang
    Lendzian, Friedhelm
    Lassmann, Günter
    Protein thiyl directly observed by EPR spectroscopy2002In: Arcives of Biochemistry & Biophysics, Vol. 403, p. 141-144Article in journal (Refereed)
  • 50.
    Langel, Ülo
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Cravatt, Benjamin F.
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Land, Tiit
    Niessen, Sherry
    Zorko, Matjaz
    Introduction to peptides and proteins2010 (ed. 1)Book (Refereed)
123 1 - 50 of 120
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